Ghrelin-Mediated Hippocampal Neurogenesis: Implications for Health and Disease

Trends in Endocrinology & Metabolism Special Issue: Advanced Themes in Endocrinology

Review

Ghrelin-Mediated Hippocampal Neurogenesis: Implications for Health and Disease Luke Buntwal,1,3 Martina Sassi,1,3 Alwena H. Morgan,1 Zane B. Andrews,2 and Jeffrey S. Davies1,* There is a close relationship between cognition and nutritional status, however, the mechanisms underlying this relationship require elucidation. The stomach hormone, ghrelin, which is released during food restriction, provides a link between circulating energy state and adaptive brain function. The maintenance of such homeostatic systems is essential for an organism to thrive and survive, and accumulating evidence points to ghrelin being key in promoting adult hippocampal neurogenesis and memory. Aberrant neurogenesis is linked to cognitive decline in ageing and neurodegeneration. Therefore, identifying endogenous metabolic factors that regulate new adult-born neurone formation is an important objective in understanding the link between nutritional status and central nervous system (CNS) function. Here, we review current developments in our understanding of ghrelin’s role in regulating neurogenesis and memory function.

Ghrelin The gut-derived hormone, ghrelin, was identified in 1999 as the natural ligand of the growth hormone secretagogue receptor 1a (GHS-R1a) (see Glossary) that stimulates the release of growth hormone (GH) from the pituitary gland [1]. An early discovery was its ability to stimulate feeding [2], resulting in the ‘hunger hormone’ name-tag. Ghrelin has subsequently been found to have numerous functions, including regulating gastric acid secretion and motility [3], pancreatic cell proliferation and apoptosis [4], sleep [5], and cardiovascular function [6]. More recently, ghrelin has been linked to central nervous system (CNS) function, including protecting neurones [7,8], regulating mood [9], and promoting neural plasticity via adult hippocampal neurogenesis (AHN) [10–12]. Ghrelin is predominantly produced by oxyntic cells of the stomach (as illustrated in Figure 1), with lower amounts generated in the pancreas [13], kidney [14], and placenta [15]. There are reports of low-level ghrelin mRNA and peptide expression in the hypothalamus [16]; however, subsequent studies unequivocally demonstrate that ghrelin is not produced in the brain [17,18]. Thus, ghrelin in the CNS derives from a peripheral source and is consistent with the ability of ghrelin to cross the blood–brain barrier (BBB) in a metabolic state-dependent manner [19,20] and bind its receptor in the brain [21,22]. Human ghrelin, encoded by the gene GHRL, located on chromosome 3p25-26, generates the preproghrelin peptide that is enzymatically processed to a mature 28 amino acid (aa) peptide [23]. The native peptide, unacylated ghrelin (UAG) (or des-acyl ghrelin), undergoes enzymatic modification in the endoplasmic reticulum by ghrelin-O-acyl transferase (GOAT) to generate acyl-ghrelin [24,25]. This involves the addition of a medium chain fatty acid (generally octanoic acid) at Serine residue 3 (Ser3), which is essential for it to bind and activate GHS-R1a signalling. Notably, GOAT expression has been observed in the hippocampus, where it is able to acylate ghrelin locally [26]. Acyl-ghrelin can also undergo de-acylation into UAG, mediated by acyl-protein thioesterase 1 (APT1) [27] or butyrylcholinesterase [28] to regulate its activity. The expression of APT1 in the brain has not been characterised, however, butyrylcholinesterase is expressed in the hippocampus [29]. As both acyl-ghrelin and UAG can cross the BBB [21], it is therefore possible that both peptides undergo enzymatic conversion within the hippocampus in a cell-specific manner. Whilst a receptor for UAG has not been identified, it has been shown to induce genome-wide expression changes in GHS-R1a knockout mice [30], suggesting the existence of an unknown UAG receptor. GHS-R1a is a 366 aa, seven transmembrane domain, G protein-coupled receptor (GPCR) [31]. It belongs to the rhodopsin family of GPCRs, primarily interacting with Gaq/11, with activation leading

844

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11 ª 2019 Elsevier Ltd. All rights reserved.

Highlights There is a close relationship between cognitive performance and nutritional status, but the mechanisms underlying this relationship are not well understood. The hormone, ghrelin, which is released during food restriction, triggers adaptive responses to improve learning and memory by increasing the formation of new neurones in the adult brain. The birth of new neurones (neurogenesis) from neural stem cells in the adult mammalian brain is an important process involved in protecting against the age-related decline in cognitive function. Activation of the hippocampal ghrelin receptor may be a viable therapeutic approach to stimulate neurogenesis and protect against age- and disease-related cognitive decline.

1Molecular Neurobiology, Institute of Life Sciences, School of Medicine, Swansea University, SA2 8PP, UK 2Department of Physiology, Biomedical Discovery Unit, Monash University, Melbourne, Australia 3These authors contributed equally to this work.

*Correspondence: [email protected]

https://doi.org/10.1016/j.tem.2019.07.001

Trends in Endocrinology & Metabolism

to the PLCb/IP3 signalling cascade. A splice isoform of the receptor, termed GHS-R1b, is a truncated 289 aa form with only five transmembrane domains, that lacks intracellular signalling ability [32]. GHSR is expressed in several peripheral tissues, including the pancreas, lungs, myocardium, liver, intestine, and adipose tissue [33]. Expression in the brain is restricted to several specific regions, including the hypothalamus, cortex, midbrain (including the substantia nigra), pons, and medulla oblongata [34,35]. Most notably, GHS-R is highly expressed in the adult hippocampus, with initial reports of GHS-R1a mRNA expression [33,36] subsequently confirmed at the protein level [34]. More specifically, the use of genetically modified GHS-R–eGFP reporter mice demonstrated that GHS-R is most highly expressed in the dentate gyrus (DG) of the hippocampus [11,35]. This pattern of receptor localisation is thought to contribute to the regulation of a diverse array of ghrelin-related behaviours, including anxiety, stress-response, feeding, reward and learning and memory [37]. These behaviours are thought to involve distinct circuits; however, where circuitry may overlap (i.e., signalling stress- and learning-related stimuli via hippocampal circuits), distinct signalling may be partially mediated by the formation of GHS-R1a dimers with other GPCRs. For example, GHS-R forms a complex with the dopamine receptor subtype 1 (D1R), which enhanced dopamine signalling in vitro [38]. Interestingly, this led to a switch in the G protein coupled to the receptor from Gaq to Gai/o, suggesting that ghrelin is able to selectively enhance dopamine signalling in cells coexpressing these proteins. However, dimerisation is thought to occur in the absence of ghrelin, indicating allosteric regulatory processes [39] (Figure 2). GHS-R1a has also been shown to modulate serotonin signalling via dimerisation with 5-HT2C receptors [40,41] and oxytocin receptors [42], whilst GHS-R1b forms heterodimers with GHS-R1a to diminish cell surface expression [32,43]. To add further complexity, GHS-R1a exhibits high constitutive activity, at least in vitro [44–46]. One potential explanation for this phenomenon is to attune a high signalling set-point to maintain energy state, necessary for survival [47]. Indeed, emerging evidence suggests ghrelin plays a role in survival, with increased levels of circulating ghrelin during energy insufficiency acting in a homeostatic manner to defend against hypoglycaemia (for review see Mani and Zigman [48]). For example, GOAT–/– mice, which lack acyl-ghrelin, show a marked increase in hypoglycaemia-induced mortality when exposed to a prolonged 48 h fast [49]. Alongside the glycaemic adaptations induced by acyl-ghrelin during energy deficit, it regulates important behavioural and physiological adaptations, beyond the stimulation of hunger, that may also play convergent roles in survival. These adaptive responses to energy deficit involve learning and memory [11], neuroprotection [8], and mood [9,50], suggesting that acylghrelin is a key metabolic hormone linking nutritional state to improvements in CNS function. Here, we focus on the evidence for ghrelin-mediated action at a key learning brain region, the hippocampus. In particular, we discuss the most recent developments in our understanding of ghrelin’s role in regulating the generation of new adult-born hippocampal neurones.

The Hippocampus The hippocampus (Figure 3), named from the Greek ‘Hippokampos’ due to its structural resemblance to the seahorse, is a major component of the limbic system that is essential to learning and memory. Studies investigating the role of the hippocampus in learning and memory were conducted on patients by William Scoville and Brenda Milner in 1957 [51]. One patient, suffering from an incurable form of epilepsy, underwent temporal lobectomy, which successfully reduced seizures but resulted in profound global amnesia. The memory dysfunction was due to the removal of a large part of the hippocampal formation and the surrounding cortical region. Indeed, subsequent studies in model organisms revealed the essential nature of the hippocampus to episodic memory [52]. This memory system is essential for processingautobiographical memories, remembering the ‘what, where, and when’ of events, thus providing spatial and contextual information for an organism to make important decisions relating to health and survival.

Ghrelin-Mediated Hippocampal Memory Since 2002, accumulating evidence has indicated ghrelin’s involvement in hippocampal memory formation in various animal models (Table 1). Initial investigations showed that a single administration of acyl-ghrelin, either intracerebrovascular (i.c.v.) [53] or intrahippocampal (i.h.) [54], promoted memory

Glossary Acyl-ghrelin: a form of ghrelin that has been acylated by GOAT, which enables it to bind to GHSR1a. Acyl-protein thioesterase 1 (APT1): an endogenous enzyme known to de-acylate acyl-ghrelin. Allosteric regulatory process: regulation of a protein or receptor independent of its active site. Brain derived neurotrophic factor (BDNF): a growth factor in the brain, involved in cell proliferation, survival, learning, and memory. Bromodeoxyuridine (BrdU): a synthetic thymidine analogue, incorporated into DNA during replication. It can be used as a marker of proliferation during a specific timeframe. Butyrylcholinesterase: an endogenous esterase enzyme, present in the plasma, that hydrolyses many different cholinebased esters, but can also deacylate acyl-ghrelin to form unacylated ghrelin (UAG). Calorie restriction: a reduction of food intake (typically 30% less) in the absence of malnutrition. Calorie restriction mimetics: endogenous or exogenous factors that mimic the effects of calorie restriction. cAMP response element binding protein (CREB): a transcription factor regulating BDNF expression and important in neuronal plasticity and memory. Dentate gyrus (DG): a subregion of the hippocampus where neurogenesis occurs. Ghrelin-O-acyl transferase (GOAT): a member of the membrane bound O-Acyl transferase (MBOAT) family, the only enzyme known to acylate ghrelin. GluA1-containing AMPA receptors: glutamate receptor and ion channels responsible for fast excitatory synaptic transmission. Important for synaptic plasticity and long-term potentiation (LTP). AMPA, a-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid. Growth hormone secretagogue receptor (GHS-R): a G proteincoupled receptor (GPCR) also known as the ghrelin receptor. It is the only known receptor for acylghrelin. GHS-R–eGFP mice: genetically mutated mice that coexpress

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

845

Trends in Endocrinology & Metabolism

retention in rats, suggesting a consolidating effect on episodic memory. Later experiments revealed that acyl-ghrelin, when administered peripherally, enhanced hippocampal dendritic-spine synapse formation, increased long-term potentiation (LTP) (a physiological correlate of memory), and improved spatial memory [22]. Moreover, ghrelin knockout mice displayed reduced CA1 spine synapses and impaired spatial memory performance in the novel object recognition (NOR) task, which was rapidly recovered by peripheral treatment with acyl-ghrelin [22]. Electrophysiological analysis demonstrated that i.h. administration of acyl-ghrelin enhanced the excitability of the hippocampus, thereby facilitating the induction of LTP and improved memory in rats [55,56]. Moreover, i.c.v. injections of acyl-ghrelin for 2 weeks promoted hippocampal LTP and memory retention while ameliorating synaptic plasticity in a rat model of Alzheimer’s disease (AD) [57]. This is consistent with the PI3K-dependent enhancement in LTP following i.h. injection of acyl-ghrelin [58]. Subsequent molecular analysis showed that the non-peptidyl GHS-R1a agonist, MK-0677, enhanced synaptic delivery of GluA1-containing AMPA receptors, consequently increasing excitatory synaptic transmission and plasticity [59]. Furthermore, acyl-ghrelin upregulated expression of the phospho-GluN2B [60] and the NR2B [61] subunit of the NMDA receptor to enhance release of the excitatory neurotransmitter, glutamate [61]. Kern et al. showed that GHS-R1a forms a heterodimer with the dopamine receptor DRD1 in a complex with Gaq ex vivo. To investigate this in vivo, mice treated with a DRD1 agonist into the DG showed enhanced hippocampal working memory, which was blocked by co-infusion of a GHS-R1a antagonist [39]. These tests indicate that DRD1-regulated behaviours depend on interactions between DRD1 and GHS-R1a in the DG. There are several lines of evidence to support a role for acyl-ghrelin in protecting neurones, which may ultimately support improvements in cognitive function. Firstly, acyl-ghrelin rescued deficits in spatial memory in streptozotocin-induced diabetic rats [62]. More specifically, acyl-ghrelin increased cAMP response element binding protein (CREB) expression and led to the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and the promotion of brain derived neurotrophic factor (BDNF) expression in the hippocampus. Moreover, acyl-ghrelin treatment of i.h. amyloid-b injected mice, that mimic aspects of AD, rescued memory deficits [63,64], decreased neuroinflammation [64], and prevented the amyloid-b induced suppression of hippocampal LTP [64]. Similarly, in amyloid-b injected rats treated with acyl-ghrelin, there was a reduction in amyloid-b plaque deposition and attenuation of memory impairments due to increased AMP-activated protein kinase (AMPK) and glycogen synthase kinase (GSK) phosphorylation and decreased tau phosphorylation [65]. In a transgenic 5xFAD mouse model of AD, neuronal cell loss was prevented in the hippocampus of mice treated with acyl-ghrelin [66]. Notably, in the senescent SAMP8 mice, acyl-ghrelin increased baseline cognitive performance, suggesting that ghrelin signalling may preserve cognitive function in ageing [22]. Collectively, these data highlight an important role for ghrelin in hippocampal function under normal and disease conditions.

AHN The process of AHN, as depicted in Figure 3, is an ongoing form of brain plasticity that combines expansion of the neural stem cell pool (to maintain the population) and differentiation, maturation, and functional integration of these newly formed neural cells. In 1962, Altman provided the first evidence of newly formed neurones in the postnatal rat hippocampus [67], termed ‘adult hippocampal neurogenesis’ (AHN). Since then, the use of bromodeoxyuridine (BrdU), a synthetic analogue of thymidine that labels proliferating cells, has greatly facilitated studies on neurogenesis in model organisms. From the 1990s, researchers studying AHN have demonstrated the existence of new adultborn neurones in the sub-granular zone (SGZ) of the human DG [68,69] (Box 1). Notably, these nerve cells show temporal functional specificity to separate highly similar components of memories into distinct memory representations that are unique and less easily confused. This function, termed ‘pattern separation’, is dependent on a reduced threshold for action potential firing when new adult-born neurones are 4–6 weeks of age [70]. A series of elegant loss-of-function [71] and gainof-function [72] studies in mice confirmed the essential role of new adult-born neurones to pattern separation-dependent memory in mice. In addition, recent studies suggest a spatial functional specificity to neurogenesis, with new adult-born neurones in the rostral pole of the DG being important for

846

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

green fluorescent protein (GFP) with the growth hormone secretagogue receptor (GHS-R) gene. Heterodimers: a protein complex, consisting of two different proteins. Long-term potentiation (LTP): an important process in memory formation, resulting in a long-term increase in the strength of electrical activity or transmission between two neurones. Neural stem progenitor cells (NSPCs): multipotent stem cells that reside in the brain with the capacity to differentiate into any neural cell type. Novel object recognition (NOR): behaviour test of cognitive function that requires recognising novel from familiar objects. This is a hippocampal-dependent task. Passive avoidance learning (PAL): behaviour test that evaluates memory and learning in rodents with neurological disorders. The rodents learn to avoid an environment in which an aversive stimulus was previously delivered. Pattern separation: the ability to distinguish similar inputs into separate discrete outputs. SAMP8 mice: a naturally occurring mouse model of accelerated ageing with symptoms of cognitive decline. Spontaneous location recognition (SLR) task: behavioural test of cognitive function that requires the ability to discriminate similar but distinct spatial contexts. This is a hippocampal neurogenesisdependent task. Synaptic plasticity: the ability of a synapse to respond to signals, thereby enhancing or decreasing the strength of connection. Thought to be an important mechanism in learning and memory. Unacylated ghrelin (UAG): the unacylated form of ghrelin [also known as des-acyl ghrelin (DAG)]. Initially thought to be inactive as it does not activate the ghrelin receptor. Subsequent research suggests it has physiological effects independent of GHS-R. 5xFAD mice: a transgenic mouse model of Alzheimer’s disease (AD) expressing mutant versions of human amyloid precursor protein (APP) [Swedish (K670N/M671L), Florida (I716V), London (V717I)], and Presenilin 1 (PSEN1) (M146L and L286V).

Trends in Endocrinology & Metabolism

Figure 1. Summary of Ghrelin Production from the Stomach. (1) Ghrelin gene (GHRL) is transcribed to preproghrelin mRNA. (2) Preproghrelin mRNA is translated to a 117 amino acid (aa) peptide in the rough endoplasmic reticulum (rER). (3) Preproghrelin peptide undergoes proteolytic cleavage to proghrelin in the ER and optional acylation by ghrelin-O-acyl transferase (GOAT). (4) Prohormone convertase (PC1/3) processes proghrelin to 28 amino acid mature ghrelin. (5) Acyl or unacylated ghrelin is secreted into the circulation. Abbreviation: SP, signal peptide.

episodic memory processing, whilst AHN in the caudal DG is linked with processing anxiety and stress-related memory [73]. The number of new adult-born neurones is regulated by several extrinsic factors. For example, aerobic exercise [74] and environmental enrichment [75] were shown to increase cell proliferation and

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

847

Trends in Endocrinology & Metabolism

Figure 2. Putative Growth Hormone Secretagogue Receptor 1a (GHS-R1a) signalling pathways. (1) Canonical GHS-R1a signalling via Gaq leading to calcium release, CamKII phosphorylation, and activation of downstream targets such as cAMP response element binding protein (CREB)/brain derived neurotrophic factor (BDNF). (2) GHS-R1a dimerisation with the dopamine receptor DRD1 regulates activation in the absence of ghrelin [39]. (3) GHS-R1a dimerisation with DRD1 results in a switch of G protein-coupled Gaq to Gai/o, leading to PI3K downstream signalling [38]. (4) GHS-R1a dimerisation with GHS-R1b results in diminished cell-surface expression [43].

survival of new adult-born neurones, respectively, in rodents. Conversely, stress [76] and ageing [77] negatively impact neurogenesis, demonstrating the remarkable plasticity of the process to factors that lay outside of the brain. Notably, alternate-day feeding [78] was shown to increase the survival of new adult-born cells in the adult DG, suggesting that factors responding to reduced calorie intake may regulate the hippocampal neurogenic niche. Here, we look at the impact of calorie restriction on hippocampal function and neurogenesis.

Benefits of Calorie Restriction on Brain Function and AHN Calorie restriction, defined as a reduction in food intake without incurring malnutrition [83], increases lifespan and protects against obesity and cardiovascular disease (for review see Di Francesco et al. [84]). In addition, calorie restriction benefits the brain by preventing cognitive decline [85] and ameliorating neurodegeneration [86,87] in rodent models of disease. Remarkably, calorie restriction reduces brain atrophy in monkeys [83,88] and improves verbal memory scores in humans [89].

848

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

Trends in Endocrinology & Metabolism

Figure 3. Stages of Adult Hippocampal Neurogenesis (AHN). AHN comprises of sequential phases of neural stem progenitor cell (NSPC) proliferation and differentiation, leading to the generation of functionally integrated neurones within the hippocampal circuitry. There are distinct NSPC types: quiescent neural progenitor cells (QNPs) or ‘type-1’ NSPCs are slowly dividing and have a single radial process extending through the granular cell layer of the dentate gyrus. As such, they are referred to as radial glial-like (RGL) cells and express nestin and glial fibrillary acidic protein (GFAP). QNPs divide to produce highly proliferative type-2 transit-amplifying progenitors (TAPs) that express Sox2 and lack radial morphology. Subsequent type 2b cells can also express neuronal markers such as NeuroD1 and Prox1 (not shown), whilst type 3 neuroblasts are characterised by Dcx immunoreactivity and lack nestin expression. During the early postmitotic phase, cells undergo programmed cell death or begin to display enhanced dendritic extensions and express NeuN. During the late maturation phase (4–6 weeks of age), cells integrate into the hippocampal network and have reduced threshold for action potential firing, an important aspect of laying down similar but distinct memories (i.e., pattern separation function). Notably, growth hormone secretagogue receptor (GHS-R) expression has been characterised throughout the differentiation process and is only expressed in the mature neuronal population [11]. Abbreviation: SGZ, subgranular zone.

Delineating the molecular pathways underlying the beneficial effects of calorie restriction remains an important objective. Currently, these mechanisms are not fully understood, however, calorie restriction is known to increase the level of BDNF in the hippocampus of adult mice [90]. BDNF is crucial for the survival of neurones during development and for improving memory function [91]. More recently, calorie restriction was shown to induce hippocampal CREB in mice [92]. CREB regulates Sirtuin1 (SIRT1), an NAD+-dependent histone deacetylase that has a key role in neuronal plasticity and memory. In addition, calorie restriction (or the pharmacological activation of SIRT1) promoted SIRT1 activity in the hippocampus, leading to preserved memory functions, delayed synaptic loss, and attenuated onset of neurodegeneration [87]. While these studies show that calorie restriction can affect the molecular machinery of hippocampal cells involved in learning and memory, it does not address how these neurones sense nutritional state during calorie restriction. Moreover, the impact of these calorie restriction paradigms on hippocampal neurogenesis were not addressed; however, an increasing number of studies are starting to examine this relationship in more detail. For example, mice exposed to a very low calorie/low protein fasting mimicking diet had increased numbers of new immature DG neurones and improved cognitive performance [93]. Given that circulating acyl-ghrelin is increased during calorie restriction, we have previously investigated circulating acyl-ghrelin as a key metabolic hormone linking nutritional state with hippocampal neurogenesis and memory [11]. To determine whether calorie restriction promotes the generation of new mature adult-born hippocampal neurones and whether acyl-ghrelin signalling is required for this effect, we exposed adult wild type and GHS-R-null mice to a 30% reduction in food intake for 2 weeks. Quantification of AHN revealed that 2 weeks of calorie restriction led to a significant 52% increase in the number of new adult-born neurones, specifically in the rostral DG of wild type mice, compared

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

849

Trends in Endocrinology & Metabolism

Model Wistar rats

Sex M

age: not specified.

Methods

Outcome: cellular

Outcome: behavioural

Refs

i.c.v. of acyl-ghrelin (0.3, 1.5,

Increased memory retention

[53]

and 3 nmol/ml)

(OF, EPM, SDT).

one injection, 7 days after surgery. Wistar rats

M

age: not specified.

i.h. of acyl-ghrelin 0.3, 1.5,

Increased memory retention

and 3 nmol/ml

and anxiogenesis (EPM,

one injection, 7 days after

SDT).

[54]

surgery. Ghr–/– and Ghsr–/– mice on

i.c.v. of acyl-ghrelin (0, 50,

Increased dendritic spine

Increased learning and

c57/Bl6 background, CD-1

75, 100, 150, 200 ng per

synapses (c57/Bl6 and Ghr–/–

memory: spontaneous

mice (4 months of age), 3-

injection).

mice), LTP (CD-1 mice).

alternation plus maze task,

month- old Sprague-Dawley

s.c. of acyl-ghrelin (10 mg/

NOR, T-maze footshock

rats, SAMP8 mice

kg) or LY444711 (5 mg/kg).

avoidance, and SDT.

(4 and 12 months).

Minipump of acyl-ghrelin

M

[22]

(3.5 mg/kg). Adult rat hippocampal

ns

progenitor cells (AHPs).

Hexarelin, acyl-ghrelin and

Hexarelin and acyl-ghrelin

Unacyl-ghrelin at 1, 3, 10,

increased proliferation.

30 mM for 24 hours.

Hexarelin protected against

[95]

apoptosis and necrosis. Lister hooded rats

M

Ghrelin receptor agonists

Improved performance in

3 mg/kg (GSK894490A oral,

the NOR and Atlantis water

CP-464709-18 s.c.) for 0.5, 1,

maze tests.

[111]

2, 3, or 6 hours. 8-week-old C57Bl/6 mice

M

i.p. ghrelin (80 mg/kg) daily

Ghrelin increased the

for 8 days.

number of BrdU+ and Dcx+

[96]

cells in the GCL of the DG. Wistar rats

M

i.h. or i.c.v. of ghrelin (0.03,

Improved long-term

0.3, 3 nmol/ml). Ghrelin

memory

administered 15 min before

(SDT and EPM).

[56]

the behavioural tests. Wistar rats

M

I.h. injection of ghrelin (0.03,

Ghrelin increased NOS

Ghrelin increased long-term

0.3, 3 nmol/ ml) 30 min before

activity and LTP.

memory (SDT).

[55]

the quantification of NOS activity or immediately after training in the SDT. i.p. of acyl-ghrelin 80 mg/kg,

Ghrelin decreased

Ghrelin rescued memory

mice (3 ml of Ab oligomers).

30 min before surgery and

Ab-induced microgliosis,

deficits (assessed by

HT22 cells incubated for 24 h

daily for 7 days.

i.h. unilateral injection in ICR

M

attenuated neuronal loss,

spontaneous alternation Y

with 1 mM of Ab oligomers in

and prevented Ab oligomer-

maze task and PAL).

presence/absence

associated synaptic

of acyl-ghrelin (10–100 nM).

degeneration in mice

[63]

injected with Ab oligomers. 8-week-old streptozotocin-induced diabetic rats.

ns

i.c.v. injection of acyl-ghrelin

Increased expression of

Improved cognitive ability

(200 ng) daily for 7 days.

BDNF, CREB, and p-CREB

(assessed by MWM).

by ERK1/2 pathway in the hippocampus.

Table 1. Summary of Studies Investigating Ghrelin or Ghrelin Mimetic Treatment and Hippocampal Functiona

850

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

[62]

Trends in Endocrinology & Metabolism

Model 2-month-old Wistar rats

8–9-week-old ghrelin KO

Sex M

M

mice

Methods

Outcome: cellular

Outcome: behavioural

i.h. injection of ghrelin (1 nM)

Enhanced LTP in the DG is

Promoted spatial memory

once or for 4 days.

PI3K-dependent.

(MWM).

i.p. injections of ghrelin

Decreased PCNA+, Ki67+,

Ghrelin administration

(80 mg/kg) daily for 8 days.

BrdU+ cells in DG, decreased

reversed memory

proportion of BrdU+/DCX+

impairment in ghrelin KO

and BrdU+/NeuN+ cells in

mice (Y-maze, NOR).

Refs [58]

[99]

ghrelin KO mice, restored to WT levels by ghrelin replacement. 2–3 month-old GH-deficient

M

spontaneous dwarf rats

i.p. injections of rat acyl-

Increased PCNA+, BrdU+,

Acyl-ghrelin administration

ghrelin (80 mg/kg) daily for

and DCX+ cells.

reversed memory

8 days or

impairment in GH-deficient

acyl-ghrelin administration

spontaneous dwarf rats (Y-

(50 mg/kg per day for 28 day)

maze, NOR).

[102]

with s.c. osmotic minipumps for the Y-maze task and NOR test. Adult rat (age 43–55 days)

Cells treated with acyl-

Increased proliferation of

hippocampal NSCs

ghrelin (1 nM to 10 mM) for

NSCs by activating MEK/

48 hours.

ERK1/2, PI3K/Akt and Jak2/

[114]

STAT3 pathways. 2-month-old 5xFAD mice

8–10-week-old C57BL/

F

M

6NCrlVr mice

i.p. injections of acyl-ghrelin

Restoration of impaired Dcx,

(80 mg/kg) every 2 days for

HH3, and calretinin in 5xFAD

[66]

30 days.

to WT levels.

i.p. rat acyl-ghrelin (80 mg/

i.p. (but not intra-CA1)

Intra-CA1 infusion of ghrelin

kg) for 8 days or dorsal CA1

increased BrdU+ cell number

impairs spatial memory

infusion (8 ng in 0.5 ml) 20 min

and proportion of BrdU+/

formation (assessed by

before MWM training

DCX+ and BrdU+/NeuN+

MWM).

sessions.

cells.

[98]

Ghrelin does not affect recruitment of new neurons into spatial memory networks. Wistar rats

M

i.h. injections of ghrelin

Increased hippocampal

Increased memory

(3 nmol/ml) for 7 days.

glutamate release Increased

consolidation (via SDT).

[61]

NR2B-NMDA receptor subunit expression. 10-week-old Tg-APPSwDI

M

(C57BL/6 background) mice

Fed control diet, high

No effect on Ab plaque load

HGI diet + ghrelin agonist

glycaemic index (HGI) diet or

or microglia activation.

improved cognition (tested

HGI diet + ghrelin agonist

[110]

using MWM).

(LY444711). 3-month-old male C57BL6/J wild type mice

M

JMV2959 (2 mg/side)

GHS-R1a and DRD1 forms

Hippocampal memory

delivered (0.5 ml/side)

heteromers in a complex

increased (contextual fear

bilaterally at a rate of 0.5 ml/

with Gaq, DRD1-induced

conditioning and T-maze

2 min.

hippocampal memory is

task).

[39]

regulated by allosteric DRD1:GHS-R1a interactions. Table 1. Continued

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

851

Trends in Endocrinology & Metabolism

Model Sprague Dawley rats

Sex M

Methods

Outcome: cellular

Outcome: behavioural

Acyl-ghrelin, unacyl-ghrelin

Acyl-ghrelin decreased Ab

Acyl-ghrelin prevented

Ab injected into lateral

or saline (i.c.v. 0.5ml/h,

plaques deposition.

memory impairment (passive

ventricle

4.8 nmol/24 h) for 14 days.

Refs [65]

avoidance test and MWM) in AD rats.

Lister hooded rats

M

+

i.p. injection of acyl-ghrelin

Increased AHN (BrdU /

Enhanced pattern

(10 mg/ml) for 14 days.

NeuN+, BrdU+, Dcx+).

separation performance via

[10]

SLR. 8–10-week- old ghrelin-KO

M

mice

Every-other-day feeding for

Increased survival of new

3 months.

cells in WT mice, but not in

[94]

ghrelin-KO mice. i.c.v. injections of acyl-

Mice showed

Ameliorated memory

& WT mice treated with Ab1-

ghrelin (0.3 mg/kg) for

neuroinflammation

deficits (NOR and modified

40

7 days.

(increased number of GFAP-

Y-maze tests).

10–12-week-old ghrelin KO

F

[64]

and Iba1-positive cells in the rostral hippocampus). Acylghrelin prevented decreased LTP (induced by Ab1-40 treatment). Sprague-Dawley rats, GHS-

M

R–/– mice

Hippocampal slice culture:

Upregulation of phospho-

acyl-ghrelin (100 nM).

GluN2B subunit of the

[60]

NMDA receptor. Adult albino Wistar rats (5 ml

F&M

of Ab1–42 into LV

i.c.v. injection of acyl-ghrelin

Ameliorated Ab-induced

Enhanced memory retention

(200ng) for 2 weeks.

synaptic plasticity

and performance (PAL).

for 7 days). 3-month-old 5xFAD mice

[57]

impairment, restoring LTP. M

i.p. injection of MK-0677

Reduced accumulation of

(5 mg/kg) for 3 weeks.

Ab, neuroinflammation,

[109]

synaptic loss, and neuronal death. Increased pCREB levels in the DG. Sprague Dawley rats, 6OHDA lesion

M

i.p. injections of saline, acyl-

Acyl-ghrelin increased Dcx+

No motor difference

ghrelin (10 mg/kg, 50 mg/kg)

cell number in the DG.

between the groups treated

or ghrelin agonist JMV-2894

with acyl-ghrelin or JMV-

(160 mg/kg) for 8 weeks.

2894 compared with control.

[12]

Table 1. Continued a Abbreviations: EPM, elevated plus maze; GCL, granule cell layer; KO, knockout; LV, left ventricle; MWM, Morris water maze; NOR, novel object recognition; NOS, nitrous oxide; ns, not specified; NSC, neural stem cell; 6-OHDA, 6-hydroxydopamine; OF, open field; PAL, passive avoidance learning; s.c., subcutaneous; WT, wild type.

with ad libitum fed wild type mice. In this study, the caudal DG did not respond to the calorie restriction stimulus, suggesting that calorie restriction may be particularly relevant to rostral DG circuits underlying learning and memory. Notably, there was no increased AHN in GHS-R-null mice in either rostral or caudal pole, demonstrating that the beneficial effect of calorie restriction on AHN was mediated by GHS-R. In addition, the increase in new adult-born neurone number seemingly contributed to enhanced remote contextual fear conditioning in the calorie restricted wild type mice [11]. However, their precise contribution needs to be further examined under conditions that place greater emphasis on pattern-separation performance. Nonetheless, these findings confirm the essential role of GHS-R in mediating the beneficial effects of calorie restriction on enhancing AHN. In support of this, a 3 month every-other-day feeding protocol increased the survival of new adult-born cells in

852

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

Trends in Endocrinology & Metabolism

Box 1. Controversy over Human AHN? Several studies have confirmed the generation and important function of new adult-born mature neurones in the adult rodent hippocampus to spatial memory performance [71,72]. However, technical limitations have resulted in some contradictory studies investigating the existence of AHN in humans (i.e., prohibited use of the carcinogen, BrdU) (for review see Kempermann [79]). For example, a recent study failed to detect AHN in aged human brain [80]. The field has been hampered by the limited collection and inconsistent preservation of postmortem human brain tissue. However, an increasing number of studies demonstrate that postmortem human tissue, collected with short postmortem interval and appropriate fixation procedures, reveal the existence of new adult-born neurones within the DG. Recently, Boldrini et al. [81] showed that AHN occurred in humans in the eighth decade of life, despite a decline in the quiescent stem cell pool, angiogenesis, and neuroplasticity. Moreover, Moreno-Jime´nez et al. [82] described an improved protocol for the identification of new adultborn neurones in the human hippocampus and confirmed their presence in aged human hippocampus, which progressively decreased in AD patients. These studies demonstrate that the AHN paradigm exists in humans and that it is modulated by environmental factors (i.e., age, disease), as observed in rodent studies. These findings suggest that AHN may be modulated to prevent cognitive decline in humans.

the DG in wild type but not ghrelin-knockout mice [94], suggesting that ghrelin signalling is important for the survival of new hippocampal cells. Collectively, these studies highlight that acyl-ghrelin is a critical metabolic hormone linking peripheral nutritional state to hippocampal neurogenesis and cognitive performance. We speculate that acyl-ghrelin agonists represent putative calorie restriction mimetics (Box 2).

Acyl-Ghrelin Treatment Increases AHN As ghrelin is seemingly unique in its ability to communicate peripheral energy status to the brain via the activation of Neuropeptide Y (NPY) and Agouti-related protein (AGRP)-dependent pathways, it is not unreasonable to suggest it plays an important role in mediating the beneficial effects of calorie restriction. Here, we discuss evidence for exogenous acyl-ghrelin regulating AHN. Johansson et al. initially described the proliferative effect of ghrelin on adult rat hippocampal progenitor cells [95]. Subsequently, experiments in mice showed that daily intraperitoneal injection (i.p.) of acyl-ghrelin at supra-physiological doses (80 mg/kg) for 8 days increased the number of newly generated cells, labelled by BrdU, in the hippocampus [96]. We later examined the longer-term effects of elevated acyl-ghrelin within the physiological range, using doses that mimic 24-hour fasting (10 mg/kg/day i.p. for 14 days) [10]. The total number of both immature neurones (Dcx+) and new adult-born neurones (BrdU+/NeuN+) were significantly increased in the DG after acyl-ghrelin treatment. The increase in AHN resulted in enhanced performance in the spontaneous location recognition (SLR) task, a neurogenesis-dependent pattern separation-dependent test [97]. Notably, the behavioural test took place 1 week after the final injection of acyl-ghrelin, suggesting that it had a long-term effect to support memory. These findings are consistent with other studies reporting ghrelin-mediated increases in AHN [12,98,99]. To further examine the mechanisms of ghrelin on AHN, we increased acyl-ghrelin levels in adult GHS-R–eGFP mice, either indirectly via an overnight fast, directly via i.p. injection (1 mg/kg), or a combination of both overnight fast and acyl-ghrelin injection. Here, all three treatment paradigms induced the expression of the immediate early gene, early growth response-1 (Egr-1) in DG neurones [11]. Egr-1 expression in mature DG neurones is an important gene involved in the selection, maturation, and functional integration of new-born neurones into the DG network [100]. However, hippocampal cell proliferation was unchanged and is consistent with previous studies investigating the impact of calorie restriction on hippocampal cell proliferation [78,94]. It is likely that ghrelin supports AHN via several distinct pathways, including via stimulation of GH [1] and consequent insulin growth factor-1, both of which can enter the brain to influence cognition and neurogenesis [101]. However, in spontaneous dwarf rats, which lack circulating GH, ghrelin’s ability to enhance AHN and cognitive performance remained intact, suggesting that it can promote AHN

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

853

Trends in Endocrinology & Metabolism

Box 2. Calorie Restriction Mimetics (CRMs): A Promising Option to Treat Disease? A primary obstacle to benefiting from the myriad effects of calorie restriction is the need to adhere to diets that limit the intake of food. Furthermore, individuals diagnosed with chronic age-related neurodegenerative conditions, such as Alzheimer’s and Parkinson’s disease, often report weight loss and reduced appetite, suggesting impaired energy balance pathways that may not fully benefit from calorie restriction. Considerable effort is on-going to develop CRMs, defined as drugs or compounds that mimic the beneficial effects of calorie restriction [107]. Indeed, a similar approach is ongoing to identify exercise-mimetics [106,108]. We suggest acylghrelin mimetics as putative CRMs to enhance cognition. Recent findings identify MK-0677, a synthetic GHS-R agonist that crosses the BBB, as a potential therapeutic cognitive enhancer as it promoted the accumulation of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor on excitatory hippocampal synapses and improved long-term potentiation (LTP) [59]. Moreover, Jeong et al. [109] showed that MK-0677 ameliorated Ab deposition, neuroinflammation, and neurodegeneration in the 5xFAD mouse model of AD. In the APPSwDI mouse model of AD the GHS-R agonist, LY444711, improved spatial memory performance [110]. Other GHS-R agonists also improved memory and cognition (in particular, GSK894490A and CP-464709-18), crossed the BBB, and promoted object recognition and spatial memory [111]. Notably, the synthetic peptide GHS-R agonist, hexarelin, promoted the survival of new adult-born hippocampal cells [112], suggesting that it may promote AHN. These results provide compelling evidence that synthetic GHS-R agonists may form a novel class of CRMs. Further studies are required to determine the effect of these putative CRMs on AHN and cognition, as well as possible negative effects on metabolism, in models of ageing and disease.

independently from the somatotropic axis [102]. Additionally, ghrelin may modulate the neurogenic niche via extra-hippocampal neural circuitry. For example, GHS-R1a in the entorhinal cortex is activated by acyl-ghrelin and calorie restriction [11] and the entorhinal cortex promotes AHN [103]. Furthermore, vagal afferents, which express GHS-R1a that regulate ghrelin-mediated feeding [104], also support hippocampal BDNF expression and neurogenesis [105]. Other mechanisms, such as the phenomenon of ligand-independent GHS-R signalling [39], have not been studied in the context of neurogenesis. Collectively, current data suggests that physiological levels of acyl-ghrelin increase AHN by promoting neurone differentiation, maturation, and survival in the neurogenic niche of the DG. Further studies are necessary to examine the DG-specific, GHS-R1a-dependent, and ghrelin-independent GHS-R1a effects on AHN.

Acyl-Ghrelin Enhances AHN in Neurodegenerative Disease Models AD is an age-related neurodegenerative disorder characterised by progressive neurone loss and deterioration of cognitive function. Hippocampal neurogenesis is impaired in AD and is thought to contribute to cognitive decline. The 5xFAD transgenic mouse model of AD exhibits impaired AHN and cognition coupled with increased hippocampal Ab deposits. Choi et al. [106] used this model to demonstrate that aerobic exercise reduced Ab levels, promoted AHN, and improved learning and memory performance. Mimicking the beneficial effects of exercise using genetic and pharmacological tools also promoted AHN and cognition, albeit without reducing Ab levels. Notably, they show that suppressing AHN increased hippocampal neurone loss in 5xFAD but not wild type mice. These data suggest that AHN plays a fundamental role in the memory deficit and neurodegeneration observed in this model of AD. Notably, acyl-ghrelin treatment (i.p.) of 5xFAD mice, at a dose of 80 mg/kg every 2 days for 30 days, increased the number of cells expressing markers of immature neurones (Dcx and calretinin) without affecting Ab levels [66]. These data suggest that elevating peripheral acyl-ghrelin is sufficient to enhance neurogenesis in this model of AD. In addition, as neurogenic deficits are associated with several neurodegenerative diseases such as Parkinson’s disease and dementia with Lewy bodies, it has been suggested that increasing AHN in these conditions may ameliorate the progression in cognitive decline or even restore cognition. Further studies are warranted to determine whether acyl-ghrelin can promote the generation of new mature adult-born neurones to rescue learning and memory deficits in models of neurodegeneration. Characterisation of GHS-R expression in the brain using a GHS-R–eGFP reporter mouse [34], identified GHS-R–eGFP+/NeuN+ immunoreactive cells in the mature DG neurone granule cell

854

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

Trends in Endocrinology & Metabolism

Figure 4. Ghrelin-Mediated Mechanism(s) Regulating Adult Hippocampal Neurogenesis (AHN). The ghrelin receptor, [growth hormone secretagogue receptor (GHS-R)] (green), is highly expressed throughout the hippocampal dentate gyrus (DG) granular cell layer (GC). GHS-R is expressed in mature GC neurones but not in neural stem progenitor cells (NSPCs) that reside within the subgranular zone (SGZ) region of the DG. Non-cell autonomous pathways, including the release of soluble pro-neurogenic factors (illustrated by vesicles) and activity-induced stimulation of NSPCs (illustrated by arrows) are highlighted as putative mechanisms of acylghrelin-mediated AHN.

population [11]. These were in apposition to both type I (nestin+) and type II (Sox2+) neural stem progenitor cells (NSPCs). GHS-R–eGFP immunoreactivity was not detected in NSPCs. Furthermore, GHS-R–eGFP was not observed in proliferating (Ki67+) cells within the SGZ. These findings suggest a non-cell-autonomous cellular mechanism for acyl-ghrelin-mediated AHN, possibly via diffusible neurotrophic factors such as BDNF, which is known to promote neurogenesis [113] and enhance pattern separation memory [97]. It is important to note that Chung et al. [114] previously reported GHS-R1a expression in these nestin+ stem cells in vitro, using both Western blot and immunocytochemistry and acyl-ghrelin enhanced proliferation via ERK1/2 and Akt pathways. However, these in vitro studies were conducted without anti-GHS-R1a antibody validation on GHS-R-knockout cells or tissues, an important control considering that generating antibodies to specific GPCRs is notoriously difficult. We recently confirmed that a GHS-R1a antibody lacked specificity using brain tissue from GHS-R-null mice [115]. Moreover, cell type-specific RNA-seq data from postnatal mouse brain, including the DG, illustrates the absence of GHS-R expression within NSPCs [116], supporting our findings obtained in GHS-R–eGFP mice [11]. Together, these studies suggest that acyl-ghrelin is acting via a non-cell-autonomous mechanism to support AHN (Figure 4). (See Figure 5.)

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

855

Trends in Endocrinology & Metabolism

Key Figure

Acyl-Ghrelin Stimulates Adult Hippocampal Neurogenesis

Outstanding Questions How does ageing effect acyl-ghrelin-mediated-AHN and can acylghrelin restore AHN in aged mammals? Which cellular and molecular mechanism(s) underpin acyl-ghrelin’s pro-neurogenic effects? Are new adult-born hippocampal neurones, generated in response to acyl-ghrelin, particularly important to the formation and maintenance of memories relating to feeding behaviour? Does acyl-ghrelin-induced AHN mediate the anti-anxiety effect of calorie restriction? Obesity has a negative effect on cognitive performance; is this mediated by a reduction in acylghrelin and subsequent drop in neurogenic drive? Does UAG modulate AHN and memory via an unidentified receptor?

Figure 5. The hormone acyl-ghrelin is released from the stomach during calorie restriction and crosses the blood– brain barrier to regulate the hippocampal neurogenic niche. Activation of the acyl-ghrelin receptor, growth hormone secretagogue receptor (GHS-R), in the hippocampal dentate gyrus enhances adult neurogenesis and learning. This signalling pathway may offer a novel therapeutic approach to ameliorate age-related cognitive decline and dementia.

Concluding Remarks and Future Perspectives Together, these findings indicate that the calorie restriction-mediated increase in plasma acyl-ghrelin, with subsequent enhancements in AHN, may confer important survival advantages to an organism. For example, this gut-derived signal for hunger increases the number of new adult-born hippocampal neurones, thereby improving discrimination of similar but distinct environments. This function would enhance the ability to remember precise locations of palatable food, particularly during periods of shortage. Similarly, the enhanced ability to distinguish safe versus dangerous environments, as a result of increased neurogenesis and enhanced pattern-separation, would improve the chances of successful refeeding and survival. In humans, acyl-ghrelin enhances hippocampal signalling relevant to feeding [117,118] and with recent studies validating the existence of hippocampal neurogenesis that is impaired with age and disease [81,82] (mimicking observations in experimental animal models) it will be important to assess the role of acyl-ghrelin on pattern-separation processes in humans (see Outstanding Questions). Together, the findings extend our understanding of how adult brain plasticity is regulated by endocrine factors of nutritional state and suggest that the ageing brain is acutely sensitive to circulating factors such as ghrelin. Next, delineating the underlying mechanisms of ghrelin-mediated AHN is necessary to develop new potential treatments for age-related cognitive decline and diseases linked with impaired AHN.

856

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

Trends in Endocrinology & Metabolism

References 1. Kojima, M. et al. (1999) Ghrelin is a growthhormone-releasing acylated peptide from stomach. Nature 402, 656–660 2. Tscho¨p, M. et al. (2000) Ghrelin induces adiposity in rodents. Nature 407, 908–913 3. Masuda, Y. et al. (2000) Ghrelin stimulates gastric acid secretion and motility in rats. Biochem. Biophys. Res. Commun. 908, 905–908 4. Granata, R. et al. (2007) Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic beta-cells and human islets: involvement of 30 ,50 -cyclic adenosine monophosphate/protein kinase A, extracellular signal-regulated kinase 1/2, and phosphatidyl inosit. Endocrinology 148, 512–529 5. Szentirmai, E. et al. (2009) The preproghrelin gene is required for the normal integration of thermoregulation and sleep in mice. Proc. Natl. Acad. Sci. U. S. A. 106, 14069–14074 6. Nagaya, N. and Kangawa, K. (2003) Ghrelin improves left ventricular dysfunction and cardiac cachexia in heart failure. Curr. Opin. Pharmacol. 3, 146–151 7. Andrews, Z.B. et al. (2009) Ghrelin promotes and protects nigrostriatal dopamine function via a UCP2-dependent mitochondrial mechanism. J. Neurosci. 29, 14057–14065 8. Bayliss, J.A. et al. (2016) Ghrelin-AMPK signaling mediates the neuroprotective effects of calorie restriction in Parkinson’s disease 36, 3049–3063 9. Lutter, M. et al. (2008) The orexigenic hormone ghrelin defends against depressive symptoms of chronic stress. Nat. Neurosci. 11, 10–11 10. Kent, B.A. et al. (2015) The orexigenic hormone acylghrelin increases adult hippocampal neurogenesis and enhances pattern separation. Psychoneuroendocrinology 51, 431–439 11. Hornsby, A.K.E. et al. (2016) Short-term calorie restriction enhances adult hippocampal neurogenesis and remote fear memory in a Ghsrdependent manner. Psychoneuroendocrinology 63, 198–207 12. Elabi, O.F. et al. (2018) The impact of ghrelin on the survival and efficacy of dopaminergic fetal grafts in the 6-OHDA-lesioned rat. Neuroscience 395, 13–21 13. Date, Y. et al. (2000) Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans 141, 4255–4261 14. Kiyoshi, M. et al. (2000) Kidney produces a novel acylated peptide, ghrelin. FEBS Lett. 486, 213–216 15. Gualillo, O. et al. (2001) Ghrelin, a novel placentalderived hormone. Endocrinology 142, 788–794 16. Mondal, M.S. et al. (2005) Identification of ghrelin and its receptor in neurons of the rat arcuate nucleus 126, 55–59 17. Sakata, I. et al. (2009) Regulatory peptides characterization of a novel ghrelin cell reporter mouse. Regul. Pept. 155, 91–98 18. Furness, J.B. et al. (2011) Investigation of the presence of ghrelin in the central nervous system of the rat and mouse. Neuroscience 193, 1–9 19. Schaeffer, M. et al. (2013) Rapid sensing of circulating ghrelin by hypothalamic appetitemodifying neurons. Proc. Natl. Acad. Sci. U. S. A. 110, 1512–1517 20. Banks, W.A. et al. (2008) Effects of triglycerides, obesity, and starvation on ghrelin transport across the blood-brain barrier. Peptides 29, 2061–2065 21. Banks, W.A. (2002) Extent and direction of ghrelin transport across the blood-brain barrier is determined by its unique primary structure. J. Pharmacol. Exp. Ther. 302, 822–827

22. Diano, S. et al. (2006) Ghrelin controls hippocampal spine synapse density and memory performance. Nat. Neurosci. 9, 381–388 23. Kojima, M. and Kangawa, K. (2005) Ghrelin: structure and function. Physiol. Rev. 85, 495–522 24. Gutierrez, J. et al. (2008) Ghrelin octanoylation mediated by an orphan lipid transferase. Proc. Natl. Acad. Sci. U. S. A. 105, 6320–6325 25. Yang, J. et al. (2008) Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 132, 387–396 26. Murtuza, M.I. and Isokawa, M. (2018) Endogenous ghrelin-O-acyltransferase (GOAT) acylates local ghrelin in the hippocampus. J. Neurochem. 144, 58–67 27. Satou, M. et al. (2010) Identification and characterization of acyl-protein thioesterase 1/lysophospholipase I as a ghrelin deacylation/ lysophospholipid hydrolyzing enzyme in fetal bovine serum and conditioned medium. Endocrinology 151, 4765–4775 28. Schopfer, L.M. et al. (2015) Pure human butyrylcholinesterase hydrolyzes octanoyl ghrelin to desacyl ghrelin. Gen. Comp. Endocrinol. 224, 61–68 29. Darvesh, S. et al. (1998) Distribution of butyrylcholine sterase in the human amygdala and hippocampal formation 390, 374–390 30. Delhanty, P.J.D. et al. (2010) Unacylated ghrelin rapidly modulates lipogenic and insulin signaling pathway gene expression in metabolically active tissues of GHSR deleted mice. PLoS One 5, e11749 31. Howard, A.D. et al. (1996) A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 273, 974–977 32. Leung, P.K. et al. (2007) The truncated ghrelin receptor polypeptide (GHS-R1b) acts as a dominant-negative mutant of the ghrelin receptor. Cell. Signal. 19, 1011–1022 33. Guan, X. et al. (1997) Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues. Mol. Brain Res. 48, 23–29 34. Mani, B.K. et al. (2014) Neuroanatomical characterization of a growth hormone secretagogue receptor-green fluorescent protein reporter mouse. J. Comp. Neurol. 3666, 3644–3666 35. Mani, B.K. et al. (2017) The role of ghrelinresponsive mediobasal hypothalamic neurons in mediating feeding responses to fasting. Mol. Metab. 6, 882–896 36. Zigman, J.M. et al. (2006) Expression of ghrelin receptor mRNA in the rat and the mouse brain. J. Comp. Neurol. 494, 528–548 37. Mu¨ller, T.D. et al. (2015) Ghrelin. Mol. Metab. 4, 437–460 38. Jiang, H. et al. (2006) Ghrelin amplifies dopamine signaling by cross talk involving formation of growth hormone secretagogue receptor/dopamine receptor subtype 1 heterodimers. Mol. Endocrinol. 20, 1772–1785 39. Kern, A. et al. (2015) Hippocampal dopamine/DRD1 signaling dependent on the ghrelin receptor. Cell 163, 1176–1190 40. Schellekens, H. et al. (2013) Promiscuous dimerization of the growth hormone secretagogue receptor (GHS-R1a) attenuates ghrelin-mediated signaling. J. Biol. Chem. 288, 181–191 41. Schellekens, H. et al. (2015) Ghreli’ns orexigenic effect is modulated via a serotonin 2C receptor interaction. ACS Chem. Neurosci. 6, 1186–1197 42. Wallace Fitzsimons, S.E. et al. (2018) A ghrelin receptor and oxytocin receptor heterocomplex

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

857

Trends in Endocrinology & Metabolism

43.

44. 45. 46.

47. 48. 49.

50.

51. 52. 53. 54.

55.

56. 57.

58.

59.

60.

61.

858

impairs oxytocin mediated signalling. Neuropharmacology 152, 90–101 Chow, K.B.S. et al. (2012) The truncated ghrelin receptor polypeptide (GHS-R1b) is localized in the endoplasmic reticulum where it forms heterodimers with ghrelin receptors (GHS-R1a) to attenuate their cell surface expression. Mol. Cell. Endocrinol. 348, 247–254 Holst, B. et al. (2003) High constitutive signaling of the ghrelin receptor — identification of a potent inverse agonist 17, 2201–2210 Holst, B. et al. (2004) Common structural basis for constitutive activity of the ghrelin receptor family. J. Biol. Chem. 279, 53806–53817 Damian, M. et al. (2012) High constitutive activity is an intrinsic feature of ghrelin receptor protein: a study with a functional monomeric GHS-R1a receptor reconstituted in lipid discs. J. Biol. Chem. 287, 3630–3641 Yanagi, S. et al. (2018) The homeostatic force of ghrelin. Cell Metab. 27, 786–804 Mani, B.K. and Zigman, J.M. (2017) Ghrelin as a survival hormone. Trends Endocrinol. Metab. 28, 843–854 Zhao, T.-J. et al. (2010) Ghrelin O-acyltransferase (GOAT) is essential for growth hormone-mediated survival of calorie-restricted mice. Proc. Natl. Acad. Sci. U. S. A. 107, 7467–7472 Walker, A.K. et al. (2015) The P7C3 class of neuroprotective compounds exerts antidepressant efficacy in mice by increasing hippocampal neurogenesis. Mol. Psychiatry 20, 500–508 Scoville, W.B. and Milner, B. (1957) Loss of recent memory after bilateral hippocampal lesions. J Neurol Neurosurg Psychiat 20, 11–21 Burgess, N. et al. (2002) The human hippocampus and spatial and episodic memory 35, 625–641 Carlini, V.P. et al. (2002) Ghrelin increases anxietylike behavior and memory retention in rats. Biochem. Biophys. Res. Commun. 299, 739–743 Carlini, V.P. et al. (2004) Differential role of the hippocampus, amygdala, and dorsal raphe nucleus in regulating feeding, memory, and anxiety-like behavioral responses to ghrelin. Biochem. Biophys. Res. Commun. 313, 635–641 Carlini, V.P. et al. (2010) Ghrelin induced memory facilitation implicates nitric oxide synthase activation and decrease in the threshold to promote LTP in hippocampal dentate gyrus. Physiol. Behav. 101, 117–123 Carlini, V.P. et al. (2010) Ghrelin and memory: differential effects on acquisition and retrieval. Peptides 31, 1190–1193 Eslami, M. et al. (2018) Chronic ghrelin administration restores hippocampal long-term potentiation and ameliorates memory impairment in rat model of Alzheimer’s disease. Hippocampus 28, 724–734 Chen, L. et al. (2011) Local infusion of ghrelin enhanced hippocampal synaptic plasticity and spatial memory through activation of phosphoinositide 3-kinase in the dentate gyrus of adult rats. Eur. J. Neurosci. 33, 266–275 Ribeiro, L.F. et al. (2014) Ghrelin triggers the synaptic incorporation of AMPA receptors in the hippocampus. Proc. Natl. Acad. Sci. U. S. A. 111, E149–E158 Berrout, L. and Isokawa, M. (2018) Ghrelin upregulates the phosphorylation of the GluN2B subunit of the NMDA receptor by activating GHSR1a and Fyn in the rat hippocampus. Brain Res. 1678, 20–26 Ghersi, M.S. et al. (2015) Ghrelin increases memory consolidation through hippocampal mechanisms dependent on glutamate release and NR2B-

62. 63.

64.

65.

66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76.

77.

78.

79. 80. 81. 82.

subunits of the NMDA receptor. Psychopharmacology 232 Ma, L.-Y. et al. (2011) Ghrelin-attenuated cognitive dysfunction in streptozotocin-induced diabetic rats. Alzheimer Dis. Assoc. Disord. 25, 352–363 Moon, M. et al. (2011) Ghrelin ameliorates cognitive dysfunction and neurodegeneration in intrahippocampal amyloid-b1-42 oligomer-injected mice. J. Alzheimers Dis. 23, 147–159 Santos, V.V. et al. (2017) Acyl ghrelin improves cognition, synaptic plasticity deficits and neuroinflammation following amyloid beta (Ab1-40) administration in mice. J. Neuroendocrinol. Published online May 2017. https://doi.org/10. 1111/jne.12476 Kang, S. et al. (2015) Central acylated ghrelin improves memory function and hippocampal AMPK activation and partly reverses the impairment of energy and glucose metabolism in rats infused with b-amyloid. Peptides 71, 84–93 Moon, M. et al. (2014) Impaired hippocampal neurogenesis and its enhancement with ghrelin in 5XFADl. J. Alzheimers Dis. 41, 233–241 Altman, J. (1962) Are new neurons formed in the brains of adult mammals? Science 135, 1127– 1128 Eriksson, P.S. et al. (1998) Neurogenesis in the adult human hippocampus. Nat. Med. 4, 1313–1317 Spalding, K.L. et al. (2013) Dynamics of hippocampal neurogenesis in adult humans. Cell 153, 1219–1227 Ge, S. et al. (2007) A critical period for enhanced synaptic plasticity in newly generated neurons of the adult brain. Neuron 54, 559–566 Clelland, C.D. et al. (2009) A functional role for adult hippocampal neurogenesis in spatial pattern separation. Science 325, 210–213 Sahay, A. et al. (2011) Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation. Nature 472, 466–470 Kheirbek, M. (2011) Dorsal vs ventral hippocampal neurogenesis: implications for cognition and mood. Neuropsychopharmacology 36, 372–373 van Praag, H. et al. (1999) Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat. Neurosci. 2, 266–270 Kempermann, G. et al. (1997) More hippocampal neurons in adult mice living in an enriched environment. Nature 386, 493–495 Gould, E. et al. (1998) Proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress. Proc. Natl. Acad. Sci. U. S. A. 95, 3168–3171 Kuhn, H.G. et al. (1996) Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J. Neurosci. 16, 2027–2033 Lee, J. et al. (2000) Dietary restriction increases the number of newly generated neural cells, and induces BDNF expression, in the dentate gyrus of rats. J. Mol. Neurosci. 15, 99–108 Kempermann, G. et al. (2018) Human adult neurogenesis: evidence and remaining questions. Cell Stem Cell 23, 25–30 Sorrells, S.F. et al. (2018) Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults. Nature 555, 377–381 Boldrini, M. et al. (2018) Human hippocampal neurogenesis persists throughout aging. Cell Stem Cell 22, 589–599 Moreno-Jime´nez, E.P. et al. (2019) Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer’s disease. Nat. Med. 25, 554–560

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

Trends in Endocrinology & Metabolism

83. Mattison, J.A. et al. (2017) Calorie restriction improves health and survival of rhesus monkeys. Nat. Commun. 8, 1–12 84. Francesco, Di et al. (2018) A time to fast. Science 365, 770–775 85. Halagappa, V.K.M. et al. (2007) Intermittent fasting and caloric restriction ameliorate age-related behavioral deficits in the triple-transgenic mouse model of Alzheimer’s disease. Neurobiol. Dis. 26, 212–220 86. Maswood, N. et al. (2004) Caloric restriction increases neurotrophic factor levels and attenuates neurochemical and behavioral deficits in a primate model of Parkinson’s disease. Proc. Natl. Acad. Sci. U. S. A. 101, 18171–18176 87. Gra¨ff, J. et al. (2013) A dietary regimen of caloric restriction or pharmacological activation of SIRT1 to delay the onset of neurodegeneration. J. Neurosci. 33, 8951–8960 88. Colman, R.J. et al. (2009) Caloric restriction delays disease onset and mortality in rhesus monkeys. Science 373, 201–204 89. Witte, A.V. et al. (2009) Caloric restriction improves memory in elderly humans. Proc. Natl. Acad. Sci. U. S. A. 106, 1255–1260 90. Lee, J. et al. (2002) Dietary restriction enhances neurotrophin expression and neurogenesis in the hippocampus of adult mice. J. Neurochem. 80, 539–547 91. Czuba, E. et al. (2018) BDNF: a key factor with multipotent impact on brain signaling and synaptic plasticity 38, 579–593 92. Fusco, S. et al. (2012) A role for neuronal cAMP responsive-element binding (CREB)-1 in brain responses to calorie restriction. Proc. Natl. Acad. Sci. U. S. A. 109, 621–626 93. Brandhorst, S. et al. (2015) A periodic diet that mimics fasting promotes multi-system regeneration, enhanced cognitive performance, and healthspan. Cell Metab. 22, 86–99 94. Kim, Y. et al. (2015) Ghrelin is required for dietary restriction-induced enhancement of hippocampal neurogenesis: lessons from ghrelin knockout mice. Endocr. J. 62, 269–275 95. Johansson, I. et al. (2008) Proliferative and protective effects of growth hormone secretagogues on adult rat hippocampal progenitor cells. Endocrinol. 149, 2191–2199 96. Moon, M. et al. (2009) Ghrelin regulates hippocampal neurogenesis in adult mice. Endocr. J. 56, 525–531 97. Bekinschtein, P. et al. (2013) BDNF in the dentate gyrus is required for consolidation of ‘‘patternseparated’’ memories. Cell Rep. 5, 759–768 98. Zhao, Z. et al. (2014) Ghrelin administration enhances neurogenesis but impairs spatial learning and memory in adult mice. Neuroscience 257, 175–185 99. Li, E. et al. (2013) Ghrelin directly stimulates adult hippocampal neurogenesis: implications for learning and memory. Endocr. J. 60, 781–789 100. Veyrac, A. et al. (2013) Zif268/egr1 gene controls the selection, maturation and functional integration of adult hippocampal newborn neurons by learning. Proc. Natl. Acad. Sci. U. S. A. 110, 7062–7067

101. Aberg, M.A. et al. (2000) Peripheral infusion of IGF-I selectively induces neurogenesis in the adult rat hippocampus. J. Neurosci. 20, 2896–2903 102. Li, E. et al. (2013) Ghrelin-induced hippocampal neurogenesis and enhancement of cognitive function are mediated independently of GH/IGF-1 axis: lessons from the spontaneous dwarf rats. Endocr. J. 60, 1065–1075 103. Stone, S.S.D. et al. (2011) Stimulation of entorhinal cortex promotes adult neurogenesis and facilitates spatial memory. J. Neurosci. 31, 13469–13484 104. Date, Y. et al. (2002) The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats. Gastroenterology 123, 1120–1128 105. O’Leary, O.F. et al. (2018) The vagus nerve modulates BDNF expression and neurogenesis in the hippocampus. Eur. Neuropsychopharmacol. 28, 307–316 106. Choi, S.H. et al. (2018) Combined adult neurogenesis and BDNF mimic exercise effects on cognition in an Alzheimer’s mouse model. Science 361, eaan8821 107. Madeo, F. et al. (2019) Caloric restriction mimetics against age-associated disease: targets, mechanisms, and therapeutic potential. Cell Metab. 29, 592–610 108. Li, S. and Laher, I. (2015) Exercise pills: at the starting line. Trends Pharmacol. Sci. 36, 906–917 109. Jeong, Y.O. et al. (2018) Mk-0677, a ghrelin agonist, alleviates amyloid beta-related pathology in 5XFAD mice, an animal model of Alzheimer’s disease. Int. J. Mol. Sci. 19, E1800 110. Kunath, N. et al. (2015) Ghrelin agonist does not foster insulin resistance but improves cognition in an Alzheimer’s disease mouse model. Sci. Rep. 5, 11452 111. Atcha, Z. et al. (2009) Cognitive enhancing effects of ghrelin receptor agonists. Psychopharmacology 206 112. Barlind, A. et al. (2010) Growth hormone & IGF research the growth hormone secretagogue hexarelin increases cell proliferation in neurogenic regions of the mouse hippocampus. Growth Hormon. IGF Res. 20, 49–54 113. Ma, D.K. et al. (2009) Neuronal activity-induced Gadd45b promotes epigenetic DNA demethylation and adult neurogenesis. Science 323, 1074–1077 114. Chung, H. et al. (2013) Multiple signaling pathways mediate ghrelin-induced proliferation of hippocampal neural stem cells. J. Endocrinol. 218, 49–59 115. Ratcliff, M. et al. (2019) Calorie restriction activates new adult born olfactory-bulb neurones in a ghrelindependent manner but acyl-ghrelin does not enhance sub-ventricular zone neurogenesis. J. Neuroendocrinol. 31, e12755 116. Zeisel, A. et al. (2018) Molecular architecture of the mouse nervous system. Cell 174, 999–1014 117. Malik, S. et al. (2008) Ghrelin modulates brain activity in areas that control appetitive behavior. Cell Metab. 7, 400–409 118. Han, J.E. et al. (2018) Ghrelin enhances food odor conditioning in healthy humans: an fMRI study. Cell Rep. 25, 2643–2652

Trends in Endocrinology & Metabolism, November 2019, Vol. 30, No. 11

859